PROJECT SUMMARY/ABSTRACT Clostridium difficile is an important pathogen capable of causing disease in animals and humans. The organism possesses genes that encode for the production of toxins. These toxins are the main virulence factor for C. difficile and are capable of causing pseudomembranous colitis and sometimes death. The targeting of these genes for amplification and detection by a real-time PCR assay allows for faster diagnosis of disease and treatment. The specific aim of validating a multiplex, real-time PCR assay for the detection of A and B toxins from Clostridium difficile is to improve laboratory detection and turnaround time in samples from infected animals and contaminated food/feed. A multiplex, real-time PCR assay is capable of detecting multiple sequences in a limited time by fragment amplification as opposed to traditional culture methods that require the growth and isolation of Clostridium difficile followed by toxin production induction and confirmation of the presence of A and B toxins. These culture methods can take days to weeks to complete while a multiplex PCR assay is performed within hours. The rapid turnaround time of the PCR assay and the ability to batch testing increases the number of assays that can be performed at one time, increasing laboratory surge testing capacity in the face of an outbreak. The PCR assay can also be used to detect the presence of toxin in multiple matrices so several sample types can be tested at the same time. Once the assay is validated for use at PADLS New Bolton Center, the longer-term objective is to utilize the assay for both FDA Vet-LIRN and Food Emergency Response Network (FERN) investigations and routine diagnostic submissions. The currently-available test is not approved for use in all animal species and all matrices so a better method of detection is needed. The assay to be validated for this project was previously published by Houser et al 2010. Our project?s matrices will include the sample types most often submitted in routine diagnostics and typical outbreak investigations: feces, dog and cat food, livestock feed, and food products routinely consumed by humans. PCR detection results will be compared against the current gold standard, toxigenic culture, performed on the same samples. Naturally-infected/contaminated samples and spiked samples will be used for the validation.